Nucleotide sequences and corresponding polypeptides conferring modulated plant size  and biomass in plants

ABSTRACT

The present invention relates to isolated nucleic acid molecules and their corresponding encoded polypeptides able confer the trait of modulated plant size, vegetative growth, organ number, plant architecture and/or biomass in plants. The present invention further relates to the use of these nucleic acid molecules and polypeptides in making transgenic plants, plant cells, plant materials or seeds of a plant having plant size, vegetative growth, organ number, plant architecture and/or biomass that are altered with respect to wild type plants grown under similar conditions

This application is a Divisional of co-pending application Ser. No.11/298,391, filed on Dec. 8, 2005, the entire contents of which arehereby incorporated by reference and for which priority is claimed under35 U.S.C. §120.

Application Ser. No. 11/298,391, filed on Dec. 8, 2005, claims priorityunder 35 U.S.C. §119(e) on U.S. Provisional Application Nos. 60/635,115and 60/635,140 filed on Dec. 8, 2005, the entire contents of which arehereby incorporated by reference.

FIELD OF THE INVENTION

The present invention relates to isolated nucleic acid molecules andtheir corresponding encoded polypeptides able to modulate plant size,vegetative growth, organ number, architecture and/or biomass in plants.The present invention further relates to using the nucleic acidmolecules and polypeptides to make transgenic plants, plant cells, plantmaterials or seeds of a plant having modulated size, vegetative growth,organ number, architecture and/or biomass as compared to wild-typeplants grown under similar conditions.

BACKGROUND OF THE INVENTION

Plants specifically improved for agriculture, horticulture, biomassconversion, and other industries (e.g. paper industry, plants asproduction factories for proteins or other compounds) can be obtainedusing molecular technologies. As an example, great agronomic value canresult from modulating the size of a plant as a whole or of any of itsorgans or the number of any of its organs.

Similarly, modulation of the size and stature of an entire plant, or aparticular portion of a plant, allows production of plants better suitedfor a particular industry. For example, reductions in the height ofspecific crops and tree species can be beneficial by allowing easierharvesting. Alternatively, increasing height, thickness or organ numbermay be beneficial by providing more biomass useful for processing intofood, feed, fuels and/or chemicals(http://www.eere.energy.gov/biomass/publications.html). Other examplesof commercially desirable traits include increasing the length of thefloral stems of cut flowers, increasing or altering leaf size and shapeor enhancing the size of seeds and/or fruits. Changes in organ size,organ number and biomass also result in changes in the mass ofconstituent molecules such as secondary products and convert the plantsinto factories for these compounds.

Availability and maintenance of a reproducible stream of food and feedto feed people has been a high priority throughout the history of humancivilization and lies at the origin of agriculture. Specialists andresearchers in the fields of agronomy science, agriculture, cropscience, horticulture, and forest science are even today constantlystriving to find and produce plants with an increased growth potentialto feed an increasing world population and to guarantee a supply ofreproducible raw materials. The robust level of research in these fieldsof science indicates the level of importance leaders in every geographicenvironment and climate around the world place on providing sustainablesources of food, feed and energy for the population.

Manipulation of crop performance has been accomplished conventionallyfor centuries through plant breeding. The breeding process is, however,both time-consuming and labor-intensive. Furthermore, appropriatebreeding programs must be specially designed for each relevant plantspecies.

On the other hand, great progress has been made in using moleculargenetics approaches to manipulate plants to provide better crops.Through introduction and expression of recombinant nucleic acidmolecules in plants, researchers are now poised to provide the communitywith plant species tailored to grow more efficiently and produce moreproduct despite unique geographic and/or climatic environments. Thesenew approaches have the additional advantage of not being limited to oneplant species, but instead being applicable to multiple different plantspecies (1).

Despite this progress, today there continues to be a great need forgenerally applicable processes that improve forest or agricultural plantgrowth to suit particular needs depending on specific environmentalconditions. To this end, the present invention is directed toadvantageously manipulating plant size, organ number, plant architectureand/or biomass to maximize the benefits of various crops depending onthe benefit sought and the particular environment in which the crop mustgrow, characterized by expression of recombinant DNA molecules inplants. These molecules may be from the plant itself, and simplyexpressed at a higher or lower level, or the molecules may be fromdifferent plant species.

SUMMARY OF THE INVENTION

The present invention, therefore, relates to isolated nucleic acidmolecules and polypeptides and their use in making transgenic plants,plant cells, plant materials or seeds of plants having life cycles,particularly plant size, vegetative growth, organ number, plantarchitecture and/or biomass, that are altered with respect to wild-typeplants grown under similar or identical conditions.

Unless otherwise defined, all technical and scientific terms used hereinhave the same meaning as commonly understood by one of ordinary skill inthe art to which this invention belongs.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1. Amino acid sequence alignment of homologues of Lead 12/67, SEQID NO. 4. Conserved regions are enclosed in a box. A consensus sequenceis shown below the alignment.

FIG. 2. Amino acid sequence alignment of homologues of Lead 17, SEQ IDNO. 10. Conserved regions are enclosed in a box. A consensus sequence isshown below the alignment.

FIG. 3. Amino acid sequence alignment of homologues of Lead 50, SEQ IDNO. 17. Conserved regions are enclosed in a box. A consensus sequence isshown below the alignment.

FIG. 4. Amino acid sequence alignment of homologues of Lead 58, SEQ IDNO. 22. Conserved regions are enclosed in a box. A consensus sequence isshown below the alignment.

FIG. 5. Amino acid sequence alignment of homologues of Lead 13/64, SEQID NO. 27. Conserved regions are enclosed in a box. A consensus sequenceis shown below the alignment.

DETAILED DESCRIPTION OF THE INVENTION 1. The Invention

The invention of the present application may be described by, but notnecessarily limited to, the following exemplary embodiments.

The present invention discloses novel isolated nucleic acid molecules,nucleic acid molecules that interfere with these nucleic acid molecules,nucleic acid molecules that hybridize to these nucleic acid molecules,and isolated nucleic acid molecules that encode the same protein due tothe degeneracy of the DNA code. Additional embodiments of the presentapplication further include the polypeptides encoded by the isolatednucleic acid molecules of the present invention.

More particularly, the nucleic acid molecules of the present inventioncomprise: (a) a nucleotide sequence encoding an amino acid sequence thatis at least 85% identical to any one of Leads 11, 17, 50, 58, 13/64 and12/67, corresponding to SEQ ID Nos. 2, 10, 17, 22, 27 and 4,respectively, (b) a nucleotide sequence that is complementary to any oneof the nucleotide sequences according to (a), (c) a nucleotide sequenceaccording to any one of SEQ ID Nos. 1, 9, 16, 21, 26 and 3, (d) anucleotide sequence that is in reverse order of any one of thenucleotide sequences according to (c) when read in the 5′ to 3′direction, (e) a nucleotide sequence able to interfere with any one ofthe nucleotide sequences according to (a), (f) a nucleotide sequenceable to form a hybridized nucleic acid duplex with the nucleic acidaccording to any one of paragraphs (a)-(e) at a temperature from about40° C. to about 48° C. below a melting temperature of the hybridizednucleic acid duplex, and (g) a nucleotide sequence encoding any one ofamino acid sequences of Leads 11, 17, 50, 58, 13/64 and 12/67,corresponding to SEQ ID Nos. 2, 10, 17, 22, 27 and 4, respectively.

Additional embodiments of the present invention include thosepolypeptide and nucleic acid molecule sequences disclosed in SEQ ID NOS:1-30.

The present invention further embodies a vector comprising a firstnucleic acid having a nucleotide sequence encoding a plant transcriptionand/or translation signal, and a second nucleic acid having a nucleotidesequence according to the isolated nucleic acid molecules of the presentinvention. More particularly, the first and second nucleic acids may beoperably linked. Even more particularly, the second nucleic acid may beendogenous to a first organism, and any other nucleic acid in the vectormay be endogenous to a second organism. Most particularly, the first andsecond organisms may be different species.

In a further embodiment of the present invention, a host cell maycomprise an isolated nucleic acid molecule according to the presentinvention. More particularly, the isolated nucleic acid molecule of thepresent invention found in the host cell of the present invention may beendogenous to a first organism and may be flanked by nucleotidesequences endogenous to a second organism. Further, the first and secondorganisms may be different species. Even more particularly, the hostcell of the present invention may comprise a vector according to thepresent invention, which itself comprises nucleic acid moleculesaccording to those of the present invention.

In another embodiment of the present invention, the isolatedpolypeptides of the present invention may additionally comprise aminoacid sequences that are at least 85% identical to any one of Leads 11,17, 50, 58, 13/64 and 12/67, corresponding to SEQ ID Nos. 2, 10, 17, 22,27 and 4, respectively.

Other embodiments of the present invention include methods ofintroducing an isolated nucleic acid of the present invention into ahost cell. More particularly, an isolated nucleic acid molecule of thepresent invention may be contacted to a host cell under conditionsallowing transport of the isolated nucleic acid into the host cell. Evenmore particularly, a vector as described in a previous embodiment of thepresent invention, may be introduced into a host cell by the samemethod.

Methods of detection are also available as embodiments of the presentinvention. Particularly, methods for detecting a nucleic acid moleculeaccording to the present invention in a sample. More particularly, theisolated nucleic acid molecule according to the present invention may becontacted with a sample under conditions that permit a comparison of thenucleotide sequence of the isolated nucleic acid molecule with anucleotide sequence of nucleic acid in the sample. The results of suchan analysis may then be considered to determine whether the isolatednucleic acid molecule of the present invention is detectable andtherefore present within the sample.

A further embodiment of the present invention comprises a plant, plantcell, plant material or seeds of plants comprising an isolated nucleicacid molecule and/or vector of the present invention. More particularly,the isolated nucleic acid molecule of the present invention may beexogenous to the plant, plant cell, plant material or seed of a plant.

A further embodiment of the present invention includes a plantregenerated from a plant cell or seed according to the presentinvention. More particularly, the plant, or plants derived from theplant, plant cell, plant material or seeds of a plant of the presentinvention preferably has increased size (in whole or in part), increasedvegetative growth, increased organ number and/or increased biomass(sometimes hereinafter collectively referred to as increased biomass) ascompared to a wild-type plant cultivated under identical conditions.Furthermore, the transgenic plant may comprise a first isolated nucleicacid molecule of the present invention, which encodes a protein involvedin early flowering, and a second isolated nucleic acid molecule whichencodes a promoter capable of driving expression in plants, wherein theincreased biomass component and the promoter are operably linked. Morepreferably, the gene conferring increased biomass may be mis-expressedin the transgenic plant of the present invention, and the transgenicplant exhibits an increased biomass as compared to a progenitor plantdevoid of the gene, when the transgenic plant and the progenitor plantare cultivated under identical environmental conditions. In anotherembodiment of the present invention increased biomass phenotype may bedue to the inactivation of a particular sequence, using for example aninterfering RNA.

A preferred embodiment consists of a plant, plant cell, plant materialor seed of a plant according to the present invention which comprises anisolated nucleic acid molecule of the present invention, wherein theplant, or plants derived from the plant, plant cell, plant material orseed of a plant, has increased biomass as compared to a wild-type plantcultivated under identical conditions.

Another embodiment of the present invention includes methods ofenhancing biomass in plants. More particularly, these methods comprisetransforming a plant with an isolated nucleic acid molecule according tothe present invention. Preferably, the method is a method of enhancingbiomass in the transformed plant, whereby the plant is transformed witha nucleic acid molecule encoding the polypeptide of the presentinvention.

Polypeptides of the present invention include consensus sequences. Theconsensus sequences are those as shown in FIGS. 1-5.

2. Definitions

The following terms are utilized throughout this application:

Biomass: As used herein, “biomass” refers to useful biological materialincluding a product of interest, which material is to be collected andis intended for further processing to isolate or concentrate the productof interest. “Biomass” may comprise the fruit or parts of it or seeds,leaves, or stems or roots where these are the parts of the plant thatare of particular interest for the industrial purpose. “Biomass”, as itrefers to plant material, includes any structure or structures of aplant that contain or represent the product of interest.

Transformation: Examples of means by which this can be accomplished aredescribed below and include Agrobacterium-mediated transformation (ofdicots (9-10), of monocots (11-13), and biolistic methods (14)),electroporation, in planta techniques, and the like. Such a plantcontaining the exogenous nucleic acid is referred to here as a T₀ forthe primary transgenic plant and T₁ for the first generation.

Functionally Comparable Proteins or Functional Homologs: This termdescribes those proteins that have at least one functionalcharacteristic in common. Such characteristics include sequencesimilarity, biochemical activity, transcriptional pattern similarity andphenotypic activity. Typically, the functionally comparable proteinsshare some sequence similarity or at least one biochemical. Within thisdefinition, analogs are considered to be functionally comparable. Inaddition, functionally comparable proteins generally share at least onebiochemical and/or phenotypic activity.

Functionally comparable proteins will give rise to the samecharacteristic to a similar, but not necessarily the same, degree.Typically, comparable proteins give the same characteristics where thequantitative measurement due to one of the comparables is at least 20%of the other; more typically, between 30 to 40%; even more typically,between 50-60%; even more typically between 70 to 80%; even moretypically between 90 to 100% of the other.

Heterologous sequences: “Heterologous sequences” are those that are notoperatively linked or are not contiguous to each other in nature. Forexample, a promoter from corn is considered heterologous to anArabidopsis coding region sequence. Also, a promoter from a geneencoding a growth factor from corn is considered heterologous to asequence encoding the corn receptor for the growth factor. Regulatoryelement sequences, such as UTRs or 3′ end termination sequences that donot originate in nature from the same gene as the coding sequence, areconsidered heterologous to said coding sequence. Elements operativelylinked in nature and contiguous to each other are not heterologous toeach other. On the other hand, these same elements remain operativelylinked but become heterologous if other filler sequence is placedbetween them. Thus, the promoter and coding sequences of a corn geneexpressing an amino acid transporter are not heterologous to each other,but the promoter and coding sequence of a corn gene operatively linkedin a novel manner are heterologous.

Misexpression: The term “misexpression” refers to an increase or adecrease in the transcription of a coding region into a complementaryRNA sequence as compared to the wild-type. This term also encompassesexpression and/or translation of a gene or coding region or inhibitionof such transcription and/or translation for a different time period ascompared to the wild-type and/or from a non-natural location within theplant genome.

Percentage of sequence identity: As used herein, the term “percentsequence identity” refers to the degree of identity between any givenquery sequence and a subject sequence. A query nucleic acid or aminoacid sequence is aligned to one or more subject nucleic acid or aminoacid sequences using the computer program ClustalW (version 1.83,default parameters), which allows alignments of nucleic acid or proteinsequences to be carried out across their entire length (globalalignment).

ClustalW calculates the best match between a query and one or moresubject sequences, and aligns them so that identities, similarities anddifferences can be determined. Gaps of one or more residues can beinserted into a query sequence, a subject sequence, or both, to maximizesequence alignments. For fast pairwise alignment of nucleic acidsequences, the following default parameters are used: word size: 2;window size: 4; scoring method: percentage; number of top diagonals: 4;and gap penalty: 5. For multiple alignment of nucleic acid sequences,the following parameters are used: gap opening penalty: 10.0; gapextension penalty: 5.0; and weight transitions: yes. For fast pairwisealignment of protein sequences, the following parameters are used: wordsize: 1; window size: 5; scoring method: percentage; number of topdiagonals: 5; gap penalty: 3. For multiple alignment of proteinsequences, the following parameters are used: weight matrix: blosum; gapopening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps:on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gln, Glu, Arg, andLys; residue-specific gap penalties: on. The output is a sequencealignment that reflects the relationship between sequences. ClustalW canbe run, for example, at the Baylor College of Medicine Search Launchersite (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and atthe European Bioinformatics Institute site on the World Wide Web(ebi.ac.uk/clustalw).

In case of the functional homolog searches, to ensure a subject sequencehaving the same function as the query sequence, the alignment has to bealong at least 80% of the length of the query sequence so that themajority of the query sequence is covered by the subject sequence. Todetermine a percent identity between a query sequence and a subjectsequence, ClustalW divides the number of identities in the bestalignment by the number of residues compared (gap positions areexcluded), and multiplies the result by 100. The output is the percentidentity of the subject sequence with respect to the query sequence. Itis noted that the percent identity value can be rounded to the nearesttenth. For example, 78.11, 78.12, 78.13, and 78.14 are rounded down to78.1, while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to78.2.

Regulatory Regions: The term “regulatory region” refers to nucleotidesequences that, when operably linked to a sequence, influencetranscription initiation or translation initiation or transcriptiontermination of said sequence and the rate of said processes, and/orstability and/or mobility of a transcription or translation product. Asused herein, the term “operably linked” refers to positioning of aregulatory region and said sequence to enable said influence. Regulatoryregions include, without limitation, promoter sequences, enhancersequences, response elements, protein recognition sites, inducibleelements, protein binding sequences, 5′ and 3′ untranslated regions(UTRs), transcriptional start sites, termination sequences,polyadenylation sequences, and introns. Regulatory regions can beclassified in two categories, promoters and other regulatory regions.

Stringency: “Stringency,” as used herein is a function of nucleic acidmolecule probe length, nucleic acid molecule probe composition (G+Ccontent), salt concentration, organic solvent concentration andtemperature of hybridization and/or wash conditions. Stringency istypically measured by the parameter T_(m), which is the temperature atwhich 50% of the complementary nucleic acid molecules in thehybridization assay are hybridized, in terms of a temperaturedifferential from T_(m). High stringency conditions are those providinga condition of T_(m)−5° C. to T_(m)−10° C. Medium or moderate stringencyconditions are those providing T_(m)−20° C. to T_(m)−29° C. Lowstringency conditions are those providing a condition of T_(m)−40° C. toT_(m)−48° C. The relationship between hybridization conditions and T_(m)(in ° C.) is expressed in the mathematical equation:

T _(m)=81.5−16.6(log₁₀[Na⁺])+0.41(% G+C)−(600/N)  (I)

where N is the number of nucleotides of the nucleic acid molecule probe.This equation works well for probes 14 to 70 nucleotides in length thatare identical to the target sequence. The equation below, for T_(m) ofDNA-DNA hybrids, is useful for probes having lengths in the range of 50to greater than 500 nucleotides, and for conditions that include anorganic solvent (formamide):

T _(m)=81.5+16.6 log {[Na⁺]/(1+0.7[Na⁺])}+0.41(% G+C)−500/L0.63(%formamide)  (II)

where L represents the number of nucleotides in the probe in the hybrid(21). The T_(m) of Equation II is affected by the nature of the hybrid:for DNA-RNA hybrids, T_(m) is 10-15° C. higher than calculated; forRNA-RNA hybrids, T_(m) is 20-25° C. higher. Because the T_(m) decreasesabout 1° C. for each 1% decrease in homology when a long probe is used(22), stringency conditions can be adjusted to favor detection ofidentical genes or related family members.

Equation II is derived assuming the reaction is at equilibrium.Therefore, hybridizations according to the present invention are mostpreferably performed under conditions of probe excess and allowingsufficient time to achieve equilibrium. The time required to reachequilibrium can be shortened by using a hybridization buffer thatincludes a hybridization accelerator such as dextran sulfate or anotherhigh volume polymer.

Stringency can be controlled during the hybridization reaction, or afterhybridization has occurred, by altering the salt and temperatureconditions of the wash solutions. The formulas shown above are equallyvalid when used to compute the stringency of a wash solution. Preferredwash solution stringencies lie within the ranges stated above; highstringency is 5-8° C. below T_(m), medium or moderate stringency is26-29° C. below T_(m) and low stringency is 45-48° C. below T_(m).

T₀: The term “T₀” refers to the whole plant, explant or callus tissue,inoculated with the transformation medium.

T₁: The term T₁ refers to either the progeny of the T₀ plant, in thecase of whole-plant transformation, or the regenerated seedling in thecase of explant or callous tissue transformation.

T₂: The term T₂ refers to the progeny of the T₁ plant. T₂ progeny arethe result of self-fertilization or cross-pollination of a T₁ plant.

T₃: The term T₃ refers to second generation progeny of the plant that isthe direct result of a transformation experiment. T₃ progeny are theresult of self-fertilization or cross-pollination of a T₂ plant.

3. Important Characteristics of the Polynucleotides and Polypeptides ofthe Invention

The nucleic acid molecules and polypeptides of the present invention areof interest because when the nucleic acid molecules are mis-expressed(i.e., when expressed at a non-natural location or in an increased ordecreased amount relative to wild-type) they produce plants that exhibitmodulated biomass as compared to wild-type plants, as evidenced by theresults of various experiments disclosed below. This trait can be usedto exploit or maximize plant products. For example, the nucleic acidmolecules and polypeptides of the present invention are used to increasethe expression of genes that cause the plant to have modulated biomass.

Because the disclosed sequences and methods increase vegetative growth,the disclosed methods can be used to enhance biomass production. Forexample, plants that grow vegetatively have an increase biomassproduction, compared to a plant of the same species that is notgenetically modified for substantial vegetative growth. Examples ofincreases in biomass production include increases of at least 10%, atleast 20%, or even at least 50%, when compared to an amount of biomassproduction by a plant of the same species not growing vegetatively.

The life cycle of flowering plants in general can be divided into threegrowth phases: vegetative, inflorescence, and floral (late inflorescencephase). In the vegetative phase, the shoot apical meristem (SAM)generates leaves that later will ensure the resources necessary toproduce fertile offspring. Upon receiving the appropriate environmentaland developmental signals the plant switches to floral, or reproductive,growth and the SAM enters the inflorescence phase (I) and gives rise toan inflorescence with flower primordia. During this phase the fate ofthe SAM and the secondary shoots that arise in the axils of the leavesis determined by a set of meristem identity genes, some of which preventand some of which promote the development of floral meristems. Onceestablished, the plant enters the late inflorescence phase (12) wherethe floral organs are produced. If the appropriate environmental anddevelopmental signals the plant switches to floral, or reproductive,growth are disrupted, the plant will not be able to enter reproductivegrowth, therefore maintaining vegetative growth.

4. The Genes of the Invention

The polynucleotides of the present invention and the proteins expressedvia translation of these polynucleotides are set forth in the SequenceListing, specifically SEQ ID Nos. 1-30. The Sequence Listing consists offunctionally comparable proteins. Polypeptides comprised of a sequencewithin and defined by one of the consensus sequences can be utilized forthe purposes of the invention, namely to make transgenic plants withmodulated biomass.

5. Use of the Genes to Make Transgenic Plants

To use the sequences of the present invention or a combination of themor parts and/or mutants and/or fusions and/or variants of them,recombinant DNA constructs are prepared that comprise the polynucleotidesequences of the invention inserted into a vector and that are suitablefor transformation of plant cells. The construct can be made usingstandard recombinant DNA techniques (see, 16) and can be introduced intothe plant species of interest by, for example, Agrobacterium-mediatedtransformation, or by other means of transformation, for example, asdisclosed below.

The vector backbone may be any of those typically used in the field suchas plasmids, viruses, artificial chromosomes, BACs, YACs, PACs andvectors such as, for instance, bacteria-yeast shuttle vectors, lamdaphage vectors, T-DNA fusion vectors and plasmid vectors (see, 17-24).

Typically, the construct comprises a vector containing a nucleic acidmolecule of the present invention with any desired transcriptionaland/or translational regulatory sequences such as, for example,promoters, UTRs, and 3′ end termination sequences. Vectors may alsoinclude, for example, origins of replication, scaffold attachmentregions (SARs), markers, homologous sequences, and introns. The vectormay also comprise a marker gene that confers a selectable phenotype onplant cells. The marker may preferably encode a biocide resistancetrait, particularly antibiotic resistance, such as resistance to, forexample, kanamycin, bleomycin, or hygromycin, or herbicide resistance,such as resistance to, for example, glyphosate, chlorosulfuron orphosphinotricin.

It will be understood that more than one regulatory region may bepresent in a recombinant polynucleotide, e.g., introns, enhancers,upstream activation regions, transcription terminators, and inducibleelements. Thus, more than one regulatory region can be operably linkedto said sequence.

To “operably link” a promoter sequence to a sequence, the translationinitiation site of the translational reading frame of said sequence istypically positioned between one and about fifty nucleotides downstreamof the promoter. A promoter can, however, be positioned as much as about5,000 nucleotides upstream of the translation initiation site, or about2,000 nucleotides upstream of the transcription start site. A promotertypically comprises at least a core (basal) promoter. A promoter alsomay include at least one control element, such as an enhancer sequence,an upstream element or an upstream activation region (UAR). For example,a suitable enhancer is a cis-regulatory element (−212 to −154) from theupstream region of the octopine synthase (ocs) gene. Fromm et al., ThePlant Cell 1:977-984 (1989).

A basal promoter is the minimal sequence necessary for assembly of atranscription complex required for transcription initiation. Basalpromoters frequently include a “TATA box” element that may be locatedbetween about 15 and about 35 nucleotides upstream from the site oftranscription initiation. Basal promoters also may include a “CCAAT box”element (typically the sequence CCAAT) and/or a GGGCG sequence, whichcan be located between about 40 and about 200 nucleotides, typicallyabout 60 to about 120 nucleotides, upstream from the transcription startsite.

The choice of promoters to be included depends upon several factors,including, but not limited to, efficiency, selectability, inducibility,desired expression level, and cell- or tissue-preferential expression.It is a routine matter for one of skill in the art to modulate theexpression of a sequence by appropriately selecting and positioningpromoters and other regulatory regions relative to said sequence.

Some suitable promoters initiate transcription only, or predominantly,in certain cell types. For example, a promoter that is activepredominantly in a reproductive tissue (e.g., fruit, ovule, pollen,pistils, female gametophyte, egg cell, central cell, nucellus,suspensor, synergid cell, flowers, embryonic tissue, embryo sac, embryo,zygote, endosperm, integument, or seed coat) can be used. Thus, as usedherein a cell type- or tissue-preferential promoter is one that drivesexpression preferentially in the target tissue, but may also lead tosome expression in other cell types or tissues as well. Methods foridentifying and characterizing promoter regions in plant genomic DNAinclude, for example, those described in the following references:Jordano, et al., Plant Cell, 1:855-866 (1989); Bustos, et al., PlantCell, 1:839-854 (1989); Green, et al., EMBO J. 7, 4035-4044 (1988);Meier, et al., Plant Cell, 3, 309-316 (1991); and Zhang, et al., PlantPhysiology 110: 1069-1079 (1996).

Examples of various classes of promoters are described below. Some ofthe promoters indicated below are described in more detail in U.S.Patent Application Ser. Nos. 60/505,689; 60/518,075; 60/544,771;60/558,869; 60/583,691; 60/619,181; 60/637,140; 10/950,321; 10/957,569;11/058,689; 11/172,703; 11/208,308; and PCT/US05/23639. It will beappreciated that a promoter may meet criteria for one classificationbased on its activity in one plant species, and yet meet criteria for adifferent classification based on its activity in another plant species.

Other Regulatory Regions: A 5′ untranslated region (UTR) can be includedin nucleic acid constructs described herein. A 5′ UTR is transcribed,but is not translated, and lies between the start site of the transcriptand the translation initiation codon and may include the +1 nucleotide.A 3′ UTR can be positioned between the translation termination codon andthe end of the transcript. UTRs can have particular functions such asincreasing mRNA stability or attenuating translation. Examples of 3′UTRs include, but are not limited to, polyadenylation signals andtranscription termination sequences, e.g., a nopaline synthasetermination sequence.

Various promoters can be used to drive expression of the genes of thepresent invention. Nucleotide sequences of such promoters are set forthin SEQ ID NOs: 31-109. Some of them can be broadly expressing promoters,others may be more tissue preferential.

A promoter can be said to be “broadly expressing” when it promotestranscription in many, but not necessarily all, plant tissues or plantcells. For example, a broadly expressing promoter can promotetranscription of an operably linked sequence in one or more of theshoot, shoot tip (apex), and leaves, but weakly or not at all in tissuessuch as roots or stems. As another example, a broadly expressingpromoter can promote transcription of an operably linked sequence in oneor more of the stem, shoot, shoot tip (apex), and leaves, but canpromote transcription weakly or not at all in tissues such asreproductive tissues of flowers and developing seeds. Non-limitingexamples of broadly expressing promoters that can be included in thenucleic acid constructs provided herein include the p326 (SEQ ID NO:),YP0144 (SEQ ID NO:), YP0190 (SEQ ID NO:), p13879 (SEQ ID NO:), YP0050(SEQ ID NO:), p32449 (SEQ ID NO:), 21876 (SEQ ID NO:), YP0158 (SEQ IDNO:), YP0214 (SEQ ID NO:), YP0380 (SEQ ID NO:), PT0848 (SEQ ID NO:), andPTO633 (SEQ ID NO:). Additional examples include the cauliflower mosaicvirus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, thefigwort mosaic virus 34S promoter, actin promoters such as the riceactin promoter, and ubiquitin promoters such as the maize ubiquitin-1promoter. In some cases, the CaMV 35S promoter is excluded from thecategory of broadly expressing promoters.

Root-active promoters drive transcription in root tissue, e.g., rootendodermis, root epidermis, or root vascular tissues. In someembodiments, root-active promoters are root-preferential promoters,i.e., drive transcription only or predominantly in root tissue.Root-preferential promoters include the YP0128 (SEQ ID NO: 82), YP0275(SEQ ID NO: 93), PT0625 (SEQ ID NO: 36), PT0660 (SEQ ID NO: 39), PT0683(SEQ ID NO: 44), and PT0758 (SEQ ID NO: 52). Other root-preferentialpromoters include the PT0613 (SEQ ID NO: 35), PT0672 (SEQ ID NO: 41),PT0688 (SEQ ID NO: 45), and PT0837 (SEQ ID NO: 54), which drivetranscription primarily in root tissue and to a lesser extent in ovulesand/or seeds. Other examples of root-preferential promoters include theroot-specific subdomains of the CaMV 35S promoter (Lam et al., Proc.Natl. Acad. Sci. USA 86:7890-7894 (1989)), root cell specific promotersreported by Conkling et al., Plant Physiol. 93:1203-1211 (1990), and thetobacco RD2 gene promoter.

In some embodiments, promoters that drive transcription in maturingendosperm can be useful. Transcription from a maturing endospermpromoter typically begins after fertilization and occurs primarily inendosperm tissue during seed development and is typically highest duringthe cellularization phase. Most suitable are promoters that are activepredominantly in maturing endosperm, although promoters that are alsoactive in other tissues can sometimes be used. Non-limiting examples ofmaturing endosperm promoters that can be included in the nucleic acidconstructs provided herein include the napin promoter, the Arcelin-5promoter, the phaseolin gene promoter (Bustos et al., Plant Cell1(9):839-853 (1989)), the soybean trypsin inhibitor promoter (Riggs etal., Plant Cell 1(6):609-621 (1989)), the ACP promoter (Baerson et al.,Plant Mol Biol, 22(2):255-267 (1993)), the stearoyl-ACP desaturase gene(Slocombe et al., Plant Physiol 104(4):167-176 (1994)), the soybean α′subunit of β-conglycinin promoter (Chen et al., Proc Natl Acad Sci USA83:8560-8564 (1986)), the oleosin promoter (Hong et al., Plant Mol Biol34(3):549-555 (1997)), and zein promoters, such as the 15 kD zeinpromoter, the 16 kD zein promoter, 19 kD zein promoter, 22 kD zeinpromoter and 27 kD zein promoter. Also suitable are the Osgt-1 promoterfrom the rice glutelin-1 gene (Zheng et al., Mol. Cell. Biol.13:5829-5842 (1993)), the beta-amylase gene promoter, and the barleyhordein gene promoter. Other maturing endosperm promoters include theYP0092 (SEQ ID NO: 68), PT0676 (SEQ ID NO: 42), and PT0708 (SEQ ID NO:47).

Promoters that drive transcription in ovary tissues such as the ovulewall and mesocarp can also be useful, e.g., a polygalacturonidasepromoter, the banana TRX promoter, and the melon actin promoter. Othersuch promoters that drive gene expression preferentially in ovules areYP0007 (SEQ ID NO: 60), YP0111 (SEQ ID NO: 76), YP0092 (SEQ ID NO: 68),YP0103 (SEQ ID NO: 73), YP0028 (SEQ ID NO: 63), YP0121 (SEQ ID NO: 81),YP0008 (SEQ ID NO: 61), YP0039 (SEQ ID NO: 64), YP0115 (SEQ ID NO: 77),YP0119 (SEQ ID NO: 62), YP0120 (SEQ ID NO: 80) and YP0374 (SEQ ID NO:98).

In some other embodiments of the present invention, embryo sac/earlyendosperm promoters can be used in order drive transcription of thesequence of interest in polar nuclei and/or the central cell, or inprecursors to polar nuclei, but not in egg cells or precursors to eggcells. Most suitable are promoters that drive expression only orpredominantly in polar nuclei or precursors thereto and/or the centralcell. A pattern of transcription that extends from polar nuclei intoearly endosperm development can also be found with embryo sac/earlyendosperm-preferential promoters, although transcription typicallydecreases significantly in later endosperm development during and afterthe cellularization phase. Expression in the zygote or developing embryotypically is not present with embryo sac/early endosperm promoters.

Promoters that may be suitable include those derived from the followinggenes: Arabidopsis viviparous-1 (see, GenBank No. U93215); Arabidopsisatmycl (see, Urao (1996) Plant Mol. Biol., 32:571-57; Conceicao (1994)Plant, 5:493-505); Arabidopsis FIE (GenBank No. AF129516); ArabidopsisMEA; Arabidopsis FIS2 (GenBank No. AF096096); and FIE 1.1 (U.S. Pat. No.6,906,244). Other promoters that may be suitable include those derivedfrom the following genes: maize MAC1 (see, Sheridan (1996) Genetics,142:1009-1020); maize Cat3 (see, GenBank No. L05934; Abler (1993) PlantMol. Biol., 22:10131-1038). Other promoters include the followingArabidopsis promoters: YP0039 (SEQ ID NO: 64), YP0101 (SEQ ID NO: 71),YP0102 (SEQ ID NO: 72), YP0110 (SEQ ID NO: 75), YP0117 (SEQ ID NO: 78),YP0119 (SEQ ID NO: 79), YP0137 (SEQ ID NO: 83), DME, YP0285 (SEQ ID NO:94), and YP0212 (SEQ ID NO: 90). Other promoters that may be usefulinclude the following rice promoters: p530c10, pOsFIE2-2, pOsMEA,pOsYp102, and pOsYp285.

Promoters that preferentially drive transcription in zygotic cellsfollowing fertilization can provide embryo-preferential expression andmay be useful for the present invention. Most suitable are promotersthat preferentially drive transcription in early stage embryos prior tothe heart stage, but expression in late stage and maturing embryos isalso suitable. Embryo-preferential promoters include the barley lipidtransfer protein (Ltp1) promoter (Plant Cell Rep (2001) 20:647-654,YP0097 (SEQ ID NO: 70), YP0107 (SEQ ID NO: 74), YP0088 (SEQ ID NO: 67),YP0143 (SEQ ID NO: 84), YP0156 (SEQ ID NO: 86), PT0650 (SEQ ID NO: 38),PT0695 (SEQ ID NO: 46), PT0723 (SEQ ID NO: 49), PT0838 (SEQ ID NO: 55),PT0879 (SEQ ID NO: 58) and PT0740 (SEQ ID NO: 50).

Promoters active in photosynthetic tissue in order to drivetranscription in green tissues such as leaves and stems are ofparticular interest for the present invention. Most suitable arepromoters that drive expression only or predominantly such tissues.Examples of such promoters include the ribulose-1,5-bisphosphatecarboxylase (RbcS) promoters such as the RbcS promoter from easternlarch (Larix laricina), the pine cab6 promoter (Yamamoto et al., PlantCell Physiol. 35:773-778 (1994)), the Cab-1 gene promoter from wheat(Fejes et al., Plant Mol. Biol. 15:921-932 (1990)), the CAB-1 promoterfrom spinach (Lubberstedt et al., Plant Physiol. 104:997-1006 (1994)),the cab1R promoter from rice (Luan et al., Plant Cell 4:971-981 (1992)),the pyruvate orthophosphate dikinase (PPDK) promoter from corn (Matsuokaet al., Proc Natl Acad. Sci. USA 90:9586-9590 (1993)), the tobaccoLhcb1*2 promoter (Cerdan et al., Plant Mol. Biol. 33:245-255 (1997)),the Arabidopsis thaliana SUC2 sucrose-H+ symporter promoter (Truernit etal., Planta 196:564-570 (1995)), and thylakoid membrane proteinpromoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD, cab,rbcS. Other promoters that drive transcription in stems, leafs and greentissue are PT0535 (SEQ ID NO: 33), PT0668 (SEQ ID NO: 32), PT0886 (SEQID NO: 59), PR0924 (SEQ ID NO: 108), YP0144 (SEQ ID NO: 85), YP0380 (SEQID NO: 100) and PT0585 (SEQ ID NO: 34).

In some other embodiments of the present invention, inducible promotersmay be desired. Inducible promoters drive transcription in response toexternal stimuli such as chemical agents or environmental stimuli. Forexample, inducible promoters can confer transcription in response tohormones such as giberellic acid or ethylene, or in response to light ordrought. Examples of drought inedible promoters are YP0380 (SEQ ID NO:100), PT0848 (SEQ ID NO: 56), YP0381 (SEQ ID NO: 101), YP0337 (SEQ IDNO: 96), YP0337 (SEQ ID NO: 96), PT0633 (SEQ ID NO: 37), YP0374 (SEQ IDNO: 98), PT0710 (SEQ ID NO: 48), YP0356 (SEQ ID NO: 97), YP0385 (SEQ IDNO: 103), YP0396 (SEQ ID NO: 104), YP0384 (SEQ ID NO: 102), YP0384 (SEQID NO: 102), PT0688 (SEQ ID NO: 45), YP0286 (SEQ ID NO: 95), YP0377 (SEQID NO: 99), and PD1367 (SEQ ID NO: 109). Examples of promoters inducedby nitrogen are PT0863 (SEQ ID NO: 57), PT0829 (SEQ ID NO: 53), PT0665(SEQ ID NO: 40) and PT0886 (SEQ ID NO: 59). An example of a shadeinducible promoter is PR0924.

Other Promoters: Other classes of promoters include, but are not limitedto, leaf-preferential, stem/shoot-preferential, callus-preferential,guard cell-preferential, such as PT0678 (SEQ ID NO: 43), andsenescence-preferential promoters. Promoters designated YP0086 (SEQ IDNO: 66), YP0188 (SEQ ID NO: 88), YP0263 (SEQ ID NO: 92), PT0758 (SEQ IDNO: 52), PT0743 (SEQ ID NO: 51), PT0829 (SEQ ID NO: 53), YP0119 (SEQ IDNO: 79), and YP0096 (SEQ ID NO: 69), as described in theabove-referenced patent applications, may also be useful.

Alternatively, misexpression can be accomplished using a two componentsystem, whereby the first component consists of a transgenic plantcomprising a transcriptional activator operatively linked to a promoterand the second component consists of a transgenic plant that comprise anucleic acid molecule of the invention operatively linked to thetarget-binding sequence/region of the transcriptional activator. The twotransgenic plants are crossed and the nucleic acid molecule of theinvention is expressed in the progeny of the plant. In anotheralternative embodiment of the present invention, the misexpression canbe accomplished by having the sequences of the two component systemtransformed in one transgenic plant line.

Another alternative consists in inhibiting expression of abiomass-modulating polypeptide in a plant species of interest. The term“expression” refers to the process of converting genetic informationencoded in a polynucleotide into RNA through transcription of thepolynucleotide (i.e., via the enzymatic action of an RNA polymerase),and into protein, through translation of mRNA. “Up-regulation” or“activation” refers to regulation that increases the production ofexpression products relative to basal or native states, while“down-regulation” or “repression” refers to regulation that decreasesproduction relative to basal or native states.

A number of nucleic-acid based methods, including anti-sense RNA,ribozyme directed RNA cleavage, and interfering RNA (RNAi) can be usedto inhibit protein expression in plants. Antisense technology is onewell-known method. In this method, a nucleic acid segment from theendogenous gene is cloned and operably linked to a promoter so that theantisense strand of RNA is transcribed. The recombinant vector is thentransformed into plants, as described above, and the antisense strand ofRNA is produced. The nucleic acid segment need not be the entiresequence of the endogenous gene to be repressed, but typically will besubstantially identical to at least a portion of the endogenous gene tobe repressed. Generally, higher homology can be used to compensate forthe use of a shorter sequence. Typically, a sequence of at least 30nucleotides is used (e.g., at least 40, 50, 80, 100, 200, 500nucleotides or more).

Thus, for example, an isolated nucleic acid provided herein can be anantisense nucleic acid to one of the aforementioned nucleic acidsencoding a biomass-modulating polypeptide. A nucleic acid that decreasesthe level of a transcription or translation product of a gene encoding abiomass-modulating polypeptide is transcribed into an antisense nucleicacid similar or identical to the sense coding sequence of thebiomass-modulating polypeptide. Alternatively, the transcription productof an isolated nucleic acid can be similar or identical to the sensecoding sequence of a biomass-modulating polypeptide, but is an RNA thatis unpolyadenylated, lacks a 5′ cap structure, or contains anunsplicable intron.

In another method, a nucleic acid can be transcribed into a ribozyme, orcatalytic RNA, that affects expression of an mRNA. (See, U.S. Pat. No.6,423,885). Ribozymes can be designed to specifically pair withvirtually any target RNA and cleave the phosphodiester backbone at aspecific location, thereby functionally inactivating the target RNA.Heterologous nucleic acids can encode ribozymes designed to cleaveparticular mRNA transcripts, thus preventing expression of apolypeptide. Hammerhead ribozymes are useful for destroying particularmRNAs, although various ribozymes that cleave mRNA at site-specificrecognition sequences can be used. Hammerhead ribozymes cleave mRNAs atlocations dictated by flanking regions that form complementary basepairs with the target mRNA. The sole requirement is that the target RNAcontain a 5′-UG-3′ nucleotide sequence. The construction and productionof hammerhead ribozymes is known in the art. See, for example, U.S. Pat.No. 5,254,678 and WO 02/46449 and references cited therein. Hammerheadribozyme sequences can be embedded in a stable RNA such as a transferRNA (tRNA) to increase cleavage efficiency in vivo. Perriman, et al.,Proc. Natl. Acad. Sci. USA, 92(13):6175-6179 (1995); de Feyter andGaudron, Methods in Molecular Biology, Vol. 74, Chapter 43, “ExpressingRibozymes in Plants”, Edited by Turner, P.C, Humana Press Inc., Totowa,N.J. RNA endoribonucleases such as the one that occurs naturally inTetrahymena thermophila, and which have been described extensively byCech and collaborators can be useful. See, for example, U.S. Pat. No.4,987,071.

Methods based on RNA interference (RNAi) can be used. RNA interferenceis a cellular mechanism to regulate the expression of genes and thereplication of viruses. This mechanism is thought to be mediated bydouble-stranded small interfering RNA molecules. A cell responds to sucha double-stranded RNA by destroying endogenous mRNA having the samesequence as the double-stranded RNA. Methods for designing and preparinginterfering RNAs are known to those of skill in the art; see, e.g., WO99/32619 and WO 01/75164. For example, a construct can be prepared thatincludes a sequence that is transcribed into an interfering RNA. Such anRNA can be one that can anneal to itself, e.g., a double stranded RNAhaving a stem-loop structure. One strand of the stem portion of a doublestranded RNA comprises a sequence that is similar or identical to thesense coding sequence of the polypeptide of interest, and that is fromabout 10 nucleotides to about 2,500 nucleotides in length. The length ofthe sequence that is similar or identical to the sense coding sequencecan be from 10 nucleotides to 500 nucleotides, from 15 nucleotides to300 nucleotides, from 20 nucleotides to 100 nucleotides, or from 25nucleotides to 100 nucleotides. The other strand of the stem portion ofa double stranded RNA comprises an antisense sequence of thebiomass-modulating polypeptide of interest, and can have a length thatis shorter, the same as, or longer than the corresponding length of thesense sequence. The loop portion of a double stranded RNA can be from 10nucleotides to 5,000 nucleotides, e.g., from 15 nucleotides to 1,000nucleotides, from 20 nucleotides to 500 nucleotides, or from 25nucleotides to 200 nucleotides. The loop portion of the RNA can includean intron. See, e.g., WO 99/53050.

In some nucleic-acid based methods for inhibition of gene expression inplants, a suitable nucleic acid can be a nucleic acid analog. Nucleicacid analogs can be modified at the base moiety, sugar moiety, orphosphate backbone to improve, for example, stability, hybridization, orsolubility of the nucleic acid. Modifications at the base moiety includedeoxyuridine for deoxythymidine, and 5-methyl-2′-deoxycytidine and5-bromo-2′-deoxycytidine for deoxycytidine. Modifications of the sugarmoiety include modification of the 2′ hydroxyl of the ribose sugar toform 2′-O-methyl or 2′-O-allyl sugars. The deoxyribose phosphatebackbone can be modified to produce morpholino nucleic acids, in whicheach base moiety is linked to a six-membered morpholino ring, or peptidenucleic acids, in which the deoxyphosphate backbone is replaced by apseudopeptide backbone and the four bases are retained. See, forexample, Summerton and Weller, 1997, Antisense Nucleic Acid Drug Dev.,7:187-195; Hyrup et al., 1996, Bioorgan. Med. Chem., 4: 5-23. Inaddition, the deoxyphosphate backbone can be replaced with, for example,a phosphorothioate or phosphorodithioate backbone, a phosphoroamidite,or an alkyl phosphotriester backbone.

Transformation

Nucleic acid molecules of the present invention may be introduced intothe genome or the cell of the appropriate host plant by a variety oftechniques. These techniques, able to transform a wide variety of higherplant species, are well known and described in the technical andscientific literature (see, e.g., 28-29).

A variety of techniques known in the art are available for theintroduction of DNA into a plant host cell. These techniques includetransformation of plant cells by injection (30), microinjection (31),electroporation of DNA (32), PEG (33), use of biolistics (34), fusion ofcells or protoplasts (35), and via T-DNA using Agrobacterium tumefaciens(36-37) or Agrobacterium rhizogenes (38) or other bacterial hosts (39),for example.

In addition, a number of non-stable transformation methods that are wellknown to those skilled in the art may be desirable for the presentinvention. Such methods include, but are not limited to, transientexpression (40) and viral transfection (41).

Seeds are obtained from the transformed plants and used for testingstability and inheritance. Generally, two or more generations arecultivated to ensure that the phenotypic feature is stably maintainedand transmitted.

A person of ordinary skill in the art recognizes that after theexpression cassette is stably incorporated in transgenic plants andconfirmed to be operable, it can be introduced into other plants bysexual crossing. Any of a number of standard breeding techniques can beused, depending upon the species to be crossed.

The nucleic acid molecules of the present invention may be used toconfer the trait of an altered flowering time.

The nucleic acid molecules of the present invention encode appropriateproteins from any organism, but are preferably found in plants, fungi,bacteria or animals.

The methods according to the present invention can be applied to anyplant, preferably higher plants, pertaining to the classes ofAngiospermae and Gymnospermae. Plants of the subclasses of theDicotylodenae and the Monocotyledonae are particularly suitable.Dicotyledonous plants belonging to the orders of the Magniolales,Illiciales, Laurales, Piperales Aristochiales, Nymphaeales,Ranunculales, Papeverales, Sarraceniaceae, Trochodendrales,Hamamelidales, Eucomiales, Leitneriales, Myricales, Fagales,Casuarinales, Caryophyllales, Batales, Polygonales, Plumbaginales,Dilleniales, Theales, Malvales, Urticales, Lecythidales, Violales,Salicales, Capparales, Ericales, Diapensales, Ebenales, Primulales,Rosales, Fabales, Podostemales, Haloragales, Myrtales, Cornales,Proteales, Santales, Rafflesiales, Celastrales, Euphorbiales, Rhamnales,Sapindales, Juglandales, Geraniales, Polygalales, Umbellales,Gentianales, Polemoniales, Lamiales, Plantaginales, Scrophulariales,Campanulales, Rubiales, Dipsacales, and Asterales, for example, are alsosuitable. Monocotyledonous plants belonging to the orders of theAlismatales, Hydrocharitales, Najadales, Triuridales, Commelinales,Eriocaulales, Restionales, Poales, Juncales, Cyperales, Typhales,Bromeliales, Zingiberales, Arecales, Cyclanthales, Pandanales, Arales,Lilliales, and Orchidales also may be useful in embodiments of thepresent invention. Further examples include, but are not limited to,plants belonging to the class of the Gymnospermae are Pinales,Ginkgoales, Cycadales and Gnetales.

The methods of the present invention are preferably used in plants thatare important or interesting for agriculture, horticulture, biomass forbioconversion and/or forestry. Non-limiting examples include, forinstance, tobacco, oilseed rape, sugar beet, potatoes, tomatoes,cucumbers, peppers, beans, peas, citrus fruits, avocados, peaches,apples, pears, berries, plumbs, melons, eggplants, cotton, soybean,sunflowers, roses, poinsettia, petunia, guayule, cabbages, spinach,alfalfa, artichokes, sugarcane, mimosa, Servicea lespedera, corn, wheat,rice, rye, barley, sorghum and grasses such as switch grass, giant reed,Bermuda grass, Johnson grass or turf grass, millet, hemp, bananas,poplars, eucalyptus trees and conifers.

Homologues Encompassed by the Invention

It is known in the art that one or more amino acids in a sequence can besubstituted with other amino acid(s), the charge and polarity of whichare similar to that of the substituted amino acid, i.e. a conservativeamino acid substitution, resulting in a biologically/functionally silentchange. Conservative substitutes for an amino acid within thepolypeptide sequence can be selected from other members of the class towhich the amino acid belongs. Amino acids can be divided into thefollowing four groups: (1) acidic (negatively charged) amino acids, suchas aspartic acid and glutamic acid; (2) basic (positively charged) aminoacids, such as arginine, histidine, and lysine; (3) neutral polar aminoacids, such as serine, threonine, tyrosine, asparagine, and glutamine;and (4) neutral nonpolar (hydrophobic) amino acids such as glycine,alanine, leucine, isoleucine, valine, proline, phenylalanine,tryptophan, cysteine, and methionine.

Nucleic acid molecules of the present invention can comprise sequencesthat differ from those encoding a protein or fragment thereof selectedfrom the group consisting of [leads 11, 17, 50, 58, 64, and 67,nucleotides] due to the fact that the different nucleic acid sequenceencodes a protein having one or more conservative amino acid changes.

Biologically functional equivalents of the polypeptides, or fragmentsthereof, of the present invention can have about 10 or fewerconservative amino acid changes, more preferably about 7 or fewerconservative amino acid changes, and most preferably about 5 or fewerconservative amino acid changes. In a preferred embodiment of thepresent invention, the polypeptide has between about 5 and about 500conservative changes, more preferably between about 10 and about 300conservative changes, even more preferably between about 25 and about150 conservative changes, and most preferably between about 5 and about25 conservative changes or between 1 and about 5 conservative changes.

Identification of Useful Nucleic Acid Molecules and their CorrespondingNucleotide Sequences

The nucleic acid molecules, and nucleotide sequences thereof, of thepresent invention were identified by use of a variety of screens thatare predictive of nucleotide sequences that provide plants with alteredsize, vegetative growth, organ number, plant architecture and/orbiomass. One or more of the following screens were, therefore, utilizedto identify the nucleotide (and amino acid) sequences of the presentinvention.

The present invention is further exemplified by the following examples.The examples are not intended to in any way limit the scope of thepresent application and its uses.

6. Experiments Confirming the Usefulness of the Polynucleotides andPolypeptides of the Invention General Protocols Agrobacterium-MediatedTransformation of Arabidopsis

Wild-type Arabidopsis thaliana Wassilewskija (WS) plants are transformedwith Ti plasmids containing clones in the sense orientation relative tothe 35S promoter. A Ti plasmid vector useful for these constructs, CRS338, contains the Ceres-constructed, plant selectable marker genephosphinothricin acetyltransferase (PAT), which confers herbicideresistance to transformed plants.

Ten independently transformed events are typically selected andevaluated for their qualitative phenotype in the T₁ generation.

Preparation of Soil Mixture: 24L SunshineMix #5 soil (Sun GroHorticulture, Ltd., Bellevue, Wash.) is mixed with 16L Therm-O-Rockvermiculite (Therm-O-Rock West, Inc., Chandler, Ariz.) in a cement mixerto make a 60:40 soil mixture. To the soil mixture is added 2 TbspMarathon 1% granules (Hummert, Earth City, Mo.), 3 Tbsp OSMOCOTE®14-14-14 (Hummert, Earth City, Mo.) and 1 Tbsp Peters fertilizer20-20-20 (J.R. Peters, Inc., Allentown, Pa.), which are first added to 3gallons of water and then added to the soil and mixed thoroughly.Generally, 4-inch diameter pots are filled with soil mixture. Pots arethen covered with 8-inch squares of nylon netting.

Planting: Using a 60 mL syringe, 35 mL of the seed mixture is aspirated.25 drops are added to each pot. Clear propagation domes are placed ontop of the pots that are then placed under 55% shade cloth andsubirrigated by adding 1 inch of water.

Plant Maintenance: 3 to 4 days after planting, lids and shade cloth areremoved. Plants are watered as needed. After 7-10 days, pots are thinnedto 20 plants per pot using forceps. After 2 weeks, all plants aresubirrigated with Peters fertilizer at a rate of 1 Tsp per gallon ofwater. When bolts are about 5-10 cm long, they are clipped between thefirst node and the base of stem to induce secondary bolts. Dippinginfiltration is performed 6 to 7 days after clipping.

Preparation of Agrobacterium: To 150 mL fresh YEB is added 0.1 mL eachof carbenicillin, spectinomycin and rifampicin (each at 100 mg/ml stockconcentration). Agrobacterium starter blocks are obtained (96-well blockwith Agrobacterium cultures grown to an OD₆₀₀ of approximately 1.0) andinoculated one culture vessel per construct by transferring 1 mL fromappropriate well in the starter block. Cultures are then incubated withshaking at 27° C. Cultures are spun down after attaining an OD₆₀₀ ofapproximately 1.0 (about 24 hours). 200 mL infiltration media is addedto resuspend Agrobacterium pellets. Infiltration media is prepared byadding 2.2 g MS salts, 50 g sucrose, and 5 μl 2 mg/ml benzylaminopurineto 900 ml water.

Dipping Infiltration: The pots are inverted and submerged for 5 minutesso that the aerial portion of the plants are in the Agrobacteriumsuspension. Plants are allowed to grow normally and seed is collected.

High-throughput Phenotypic Screening of Misexpression Mutants: Seed isevenly dispersed into water-saturated soil in pots and placed into adark 4° C. cooler for two nights to promote uniform germination. Potsare then removed from the cooler and covered with 55% shade cloth for4-5 days. Cotyledons are fully expanded at this stage. FINALE® (SanofiAventis, Paris, France) is sprayed on plants (3 ml FINALE® diluted into48 oz. water) and repeated every 3-4 days until only transformantsremain.

Screening: Screening is routinely performed at four stages: Seedling,Rosette, Flowering, and Senescence.

-   -   Seedling—the time after the cotyledons have emerged, but before        the 3^(rd) true leaf begins to form.    -   Rosette—the time from the emergence of the 3^(rd) true leaf        through just before the primary bolt begins to elongate.    -   Flowering—the time from the emergence of the primary bolt to the        onset of senescence (with the exception of noting the flowering        time itself, most observations should be made at the stage where        approximately 50% of the flowers have opened).    -   Senescence—the time following the onset of senescence (with the        exception of “delayed senescence”, most observations should be        made after the plant has completely dried). Seeds are then        collected.

Screens: Screening for increased size, vegetative growth and/or biomassis performed by taking measurements, specifically T₂ measurements weretaken as follows:

-   -   Days to Bolt=number of days between sowing of seed and emergence        of first inflorescence.    -   Rosette Leaf Number at Bolt=number of rosette leaves present at        time of emergence of first inflorescence.    -   Rosette Area=area of rosette at time of initial inflorescence        emergence, using formula ((L×W)*3.14)/4.    -   Height=length of longest inflorescence from base to apex. This        measurement was taken at the termination of flowering/onset of        senescence.    -   Primary Inflorescence Thickness=diameter of primary        inflorescence 2.5 cm up from base. This measurement was taken at        the termination of flowering/onset of senescence.    -   Inflorescence Number=total number of unique inflorescences. This        measurement was taken at the termination of flowering/onset of        senescence.

PCR was used to amplify the cDNA insert in one randomly chosen T₂ plant.This PCR product was then sequenced to confirm the sequence in theplants.

7. Results Example 1 Lead 11; (ME01795; Ceres cDNA 5662747; clone 8161)

Ten independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation as per standardprotocol. Three events showing the strongest T₁ phenotypes were chosenfor evaluation in the T₂ generation. The T₂ growth conditions followedthe above T₁ protocol. The experimental design differed from the T₁planting in that each T₂ plant was contained with its own pot, and noherbicide selection was used. All the pots for each T₂ event werecontained within the same flat and the plants were randomly distributedwithin each flat. The controls for each set of measurements were thesegregating progeny of other T₁ events which did not contain this gene(internal controls). All analyses were done via soil-based experimentsunder long day light conditions (16 hours) in the Ceres greenhouse.

All ten events showed a variety of phenotypes different from wild-typetransgenic controls (Table 1. The most pronounced variant phenotype wasthat of reduced secondary inflorescence formation, slightly delayedflowering time, larger rosettes with more leaves, and tall, thickinflorescences.

TABLE 1 Qualitative phenotypes observed in 35S::cDNA 5662747 T₁ events

Events 01, 04, and 10 were evaluated in greater detail in the T₂generation. Fourteen individuals were sown for each event. Thetransgenic plants of all 3 events showed increased height, primaryinflorescence thickness, and delay of flowering time to a 0.01 level ofstatistical significance (Table 2). These plants also had qualitativelylarger rosettes which contained more leaves (data not shown). Allplants, noted in the table as ME01795-01, ME01795-04, or ME01795-10,were segregating progeny of the T₁ event which we had confirmed tocontain the transgene under test. All plants noted in the table as -01Control, -04 Control, or -10 Controls were segregating progeny of the T₁event which did not contain the transgene under test (internalcontrols).

One item of note in the T₂ analysis is that the reduced secondaryinflorescence formation observed in T₁ plants is no longer present in T₂plants. In addition, the delay in flowering time appears to haveincreased in severity from the T₁ to T₂ generation.

Segregation frequencies of the transgene under test suggest that eachevent contains a single insert, as determined by a Chi-square test (datanot shown).

TABLE 2 Quantitative phenotypes seen in 35S::cDNA 5662747 T₂ eventsNumber of Height Primary Inflorescence Days to Event/ControlObservations (cm) Thickness (mm) Bolt ME01795-01 8 64.3* 1.062* 29.8*-01 Control 6 48.3 1.048 24.5 ME01795-04 9 70.9* 1.065* 35.8* -04Control 5 42.4 1.047 25.8 ME01795-10 8 67.9* 1.069* 31.3* -10 Control 643.3 1.049 25.3 *significantly different from control at 0.01 level, viat-test

Summary of Results:

-   -   The ectopic expression of cDNA 5662747 with a strong        constitutive promoter (35S) results in taller plants, with        thicker inflorescences, a larger rosette, and more rosette        leaves.    -   5662747 is normally expressed in shoot and root apices,        suggesting that the encoded protein may help to regulate        meristem function.    -   The increase in plant size seen by this expression is        accompanied by a delay in flowering time, but no reduction in        fertility.    -   As the T₁ plants had a much less severe delay in flowering than        the T₂ plants, but still produced the large-plant phenotype, it        may be possible to use a promoter of different strength or with        a different spatial expression pattern with the cDNA to maintain        an increase in plant height and stem/inflorescence thickness        without any increase in flowering time.    -   It may also be a useful gene to increase root growth, given the        similar expression pattern in shoot meristems and root tip        cells.

As a result, this gene is useful to increase vegetative biomass to givean improved source:sink ratio and improved fixation of carbon to sucroseand starch. Taller inflorescences give the opportunity for more flowersand therefore more seeds. The combination of improved biomass andinflorescence stature can give a significant improvement in yield.Thicker inflorescences can prevent against “snap” against wind, rain ordrought and there was no detectable difference in fertility factors suchas silique number and seed fill.

Example 2 Lead 12/67; ME03459 and ME04358; Ceres cDNA 12337825/14296577;Clone 8490/96

Experiments were performed substantially as described in Example 1, butwith different seeds. Ten independently transformed events were selectedand evaluated for their qualitative phenotype in the T₁ generation asper standard protocol. Two events showing the most advantageous T₁phenotypes (large, late-flowering) were chosen for evaluation in the T₂generation. The T₂ growth conditions follow the above T₁ protocol. Theexperimental design differs from the T₁ planting in that each T₂ plantis contained within its own pot, and no herbicide selection is used. Allpots for each T₂ event are contained within the same flat and the plantsare randomly distributed within each flat. The controls for each set ofmeasurements are the segregating progeny of the given T₁ event which donot contain the T-DNA (internal controls). All analyses are done viasoil-based experiments under long day light conditions (16 hours) in theCeres greenhouse.

All ten events were late flowering, produced larger rosettes with moreleaves and tall, thick inflorescences compared to the controls (Table3). The transgenic “control” was a set of different 35S cDNA expressingplants which were indistinguishable from the untransformed WS wild type.

TABLE 3 Qualitative phenotypes observed in 35S::cDNA 12337825 T₁ events

Events ME03459-01 and ME03459-04 were evaluated in greater detail in theT₂ generation. Seventeen individuals were sown and observed for event01, whereas 18 individuals were sown and observed for event 04. Thetransgenic plants for both events showed increased height, increasedprimary inflorescence thickness, increased number of rosette leaves, alarger rosette, and delay of flowering time to a 0.05 level ofstatistical significance (Table 4). Both events had normal fertility.All plants noted in the table as ME03459-01 or ME03459-04 weresegregating progeny of the T₁ event which we had confirmed to containthe transgene under test. All plants noted in the table as -01 Controlor -04 Control were segregating progeny of the given T1 event, which didnot contain the transgene (internal controls).

Both events produce significantly more seeds than the control, as wouldbe expected for a typical, fertile, late flowering plant.

Event ME03459-01 exhibited the strongest as noted in Table 4. Therosette area, number of leaves, thickness of the inflorescence and daysto bolt are all greater than event -04.

Segregation frequencies of the transgene under test suggest that eachevent contains a single insert, as calculated by a Chi-square test.

TABLE 4 Quantitative phenotypes observed in 35S::cDNA 12337825 T₂ eventsNumber of Rosette Number Height Primary Inflorescence Event/ControlObservations Area (mm²) of Leaves (cm) Thickness (inches) Days to BoltME03459-01 14 7023.0* 11.0* 75.6* 0.068* 21.9* -01 Control 3 2348.5 8.052.2 0.050 19.0 ME03459-04 9 4977.7* 9.4* 68.9* 0.055* 20.8* -04 Control5 2521.1 7.5 54.0 0.051 18.1 *significantly different from control at0.05 level, via t-test

Summary of Results:

-   -   The ectopic expression of cDNA 12337825 with a strong        constitutive promoter (35S) results in taller plants, with        thicker inflorescences, a larger rosette, and increased number        of rosette leaves.    -   12337825 is normally regulated in shoot and root apices,        suggesting that the encoded protein may help to regulate        meristem function.    -   The increase in plant size observed by this expression is        accompanied by a delay in flowering time, but no reduction in        fertility.    -   It is also a useful gene to increase root growth, given the        similar expression pattern in shoot meristems and root tip        cells.

As a result, this gene is useful to increase vegetative biomass and givean improved source:sink ratio and improved fixation of carbon to sucroseand starch, and to improved yield. Taller inflorescences give theopportunity for more flowers and therefore more seeds. The combinationof improved biomass and inflorescence stature can give a significantimprovement in yield. In addition, thicker inflorescences can prevent“snap” against wind, rain or drought.

ME04358 was identified from a T₁ screen looking for mutant developmentaland morphological phenotypes. All four independent transgenic eventscontaining Clone 96 (ME04358) were screened in the T₁ generation for thepresence of mutant developmental and morphological phenotypes. Allevents were taller with larger rosettes than wildtype.

Events ME04358-01 and -02 show 3:1 and 15:1 (R:S) segregation forFinale™, respectively. Event ME04358-01 showed a Mendelian segregationof 3:1 (R:S) for Finale™ in the T₂ generation. Event ME04358-02segregated 15:1 (R:S) for Finale™ in the T₂ generation.

Both T₂ events of ME04358 show increased rosette/leaf size andinflorescence height compared to controls. Events ME04358-01 and -02were tested for the quantitative traits of Plant Size. When grown underconstant light, both events had significantly larger rosettes and tallerinflorescences with a p-value less than or equal to 0.05 (Table 5). Bothevents were also slightly, yet statistically significantly laterflowering than controls. Both events trended toward having slightlythicker inflorescences than the controls, but these data were notstatistically significant at p≦0.05. Under long-day conditions, theplants were larger than controls but had an exaggerated delay inflowering time.

TABLE 5 Analysis of Rosette Area, Primary Inflorescence Thickness, PlantHeight, and Days to Flowering for ME04358-01 & -02. Both events hadstatistically larger rosettes, taller inflorescences, and a slight delayin flowering. All values denote the mean of the population.

*Indicates statistical significance with p ≦ 0.05, via t-test

Summary of Results:

The four T₁ plants were larger and taller than the controls. EventsME04358-01 and -02 were screened.

-   -   Germination—No detectable reduction in germination rate    -   General morphology/architecture—Plants were larger than the        controls    -   Days to flowering—A statistical delay in flowering was observed        for both events    -   Rosette area 7 days post-bolting—The rosettes of both events        were larger than controls    -   Plant height—An increase in height was observed for both events    -   Fertility (silique number and seed fill)—No observable        differences between experimentals and controls.

Example 3 Lead 13/64; ME01990; Ceres cDNA 12420510/1434106; clone258241/25821

Four independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation as per standardprotocol. Two events showing the most advantageous T₁ phenotypes (largeplants with larger rosettes, more leaves and tall, thick inflorescences)were chosen for evaluation in the T₂ generation. The T₂ growthconditions follow the above T₁ protocol. The experimental design differsfrom the T₁ planting in that each T₂ plant is contained within its ownpot, and no herbicide selection is used. All pots for each T₂ event arecontained within the same flat and the plants are randomly distributedwithin each flat. The controls for each set of measurements are thesegregating progeny of the given T₁ event which do not contain the T-DNA(internal controls). All analyses are done via soil-based experimentsunder long day light conditions (16 hours) in the Ceres greenhouse.

Two of the 4 events produced larger rosettes with more leaves and tall,thick inflorescences (Table 6) compared to the controls. The transgenic“control” was a set of different 35S::cDNA expressing plants which wereindistinguishable from the untransformed WS wildtype.

TABLE 6 Qualitative phenotypes observed in 35S::cDNA 12420510 T₁ events

Events ME01990-02 and ME01990-03 were evaluated in greater detail in theT₂ generation. Eighteen individuals were sown and observed for eachevent. The transgenic plants for both events showed increased height,increased number of rosette leaves, a larger rosette, and delay offlowering time to a 0.05 level of statistical significance (Table 7).Although both events had visibly thicker inflorescences in the T1generation, only event -03—proved to be quantitatively thicker to a 0.05level of statistical significance (Table 7). Both events had normalfertility. All plants noted in the table as ME01990-02 and ME01990-03were segregating progeny of the T₁ event which we had confirmed tocontain the transgene under test. All plants noted in the table as -02Control or -03 Control were segregating progeny of given T1 events whichdid not contain the transgene (internal controls).

Both events produce significantly more seeds than the control, as wouldbe expected for a typical, fertile, late flowering plant.

Event ME01991-02 had 6 transgene-containing plants which exhibited thebeneficial phenotype and 8 transgene-containing plants which appearedwild-type (not included in the statistical analysis in Table 7). EventME01991-03 had 7 transgene-containing plants which exhibited thebeneficial phenotype and 1 transgene-containing plant which appearedwild-type. Our statistical analyses compared the internal controls tothose plants with the beneficial phenotype which contained thetransgene.

Segregation frequencies of the transgene under test suggest that eachevent contains a single insert, as calculated by a Chi-square test.

TABLE 7 Quantitative phenotypes observed in 35S::cDNA 12420510 T₂ eventsNumber of Rosette Number Height Primary Inflorescence Event/ControlObservations Area (mm²) of Leaves (cm) Thickness (inches) Days to BoltME01990-02 6 4589.4* 10.8* 59.3* 0.056 21.5* -02 Control 4 2628.0 8.854.3 0.053 19.3 ME01990-03 7 5589.0* 11.3* 57.3* 0.057* 22.9 -03 Control10 1905.2 7.9 48.6 0.048 18.9 *significantly different from control at0.05 level, via t-test

Summary of Results:

-   -   The ectopic expression of cDNA 12420510 with a strong        constitutive promoter (35S) results in taller plants, a larger        rosette, and more rosette leaves.    -   The increase in plant size seen by this expression is        accompanied by a slight delay in flowering time, but no        reduction in fertility. This would be a particularly useful gene        to employ in crop plants because flowering time is only slightly        delayed.

As a result, this gene is useful to increase vegetative biomass and givean improved source:sink ratio and improved fixation of carbon to sucroseand starch, and to improved yield. Taller inflorescences give theopportunity for more flowers and therefore more seeds. The combinationof improved biomass and inflorescence stature can give a significantimprovement in yield. In addition, thicker inflorescences can prevent“snap” against wind, rain or drought.

ME01990-01, -03; Ceres cDNA 14301106; clone 25821

T₂ plants were scored quantitatively for the previously observed PlantSize phenotype and statistical significance was determined, as perprotocol, with one exception. Twelve segregating experimentals and 12external controls were sown per flat per event.

ME01990 was identified from a T₁ screen looking for mutant developmentaland morphological phenotypes. All 4 independent transgenic eventscontaining Clone 258241 (ME01990) were screened in the T₁ generation forthe presence of mutant developmental and morphological phenotypes.Events -02 and -03 appeared larger than the rest of the population.

Both events of ME01990 show 3:1 segregation for Finale™.

Both T₂ events of ME01990 show increased rosette/leaf size andinflorescence thickness compared to controls. ME01593, containing thesame construct yielded similar results. Events ME01990-02 and -03 weretested for the quantitative traits of Plant Size. Both events hadsignificantly larger rosettes and thicker inflorescences with a p-valueless than or equal to 0.05 (Table 8). Event -03 was also slightly, yetstatistically significantly later flowering than controls. However, thedegree of flowering delay falls within the window of acceptableflowering time as negative phenotypes are defined. Both events trendedtoward being slightly taller than the controls, but these data were notstatistically significant at p<0.05. When the two events of ME01990 weregrown in constant light, they were wildtype in appearance.

In an unrelated experiment, events of ME01593 (also 35S::258241) weretested for the noted quantitative plant size traits under long dayconditions. The results of this experiment were equivalent to those ofME01990. ME01593 events had larger rosettes, thicker inflorescences, andwere delayed in time to flowering.

From the T₁ analyses of the degree of biomass and late flowering, 14 ofthe large late flowering lines were tested in the T₂ generation underlong day and constant light conditions. Most of the 14 lines wereseverely delayed in flowering under long day and constant lightconditions, but several, while delayed in flowering under long dayconditions, exhibited a wildtype phenotype under constant light. Assuch, ME01990 is a late flowering line that has an increased biomass butless than four days delay in flowering under long day conditions.

TABLE 8 Analysis of Rosette Area, Primary Inflorescence Thickness, PlantHeight, and Day to Flowering for ME01990-02 & -03. Both events hadstatistically larger rosettes and thicker inflorescences. All valuesdenote the mean of the population.

*Indicates statistical significance with p ≦ 0.05, via t-test

Events -02 and -03 of ME01990 were screened. Twelve segregatingtransformants from each event were screened instead of 18, and 12external wild-type controls were screened instead of 6.

Summary of Results:

-   -   Germination—no detectable reduction in germination rate    -   General morphology/architecture—plants were larger than the        controls    -   Days to flowering—A statistical delay in flowering was observed        for Event -03 but not for -02.    -   Rosette area 7 days post-bolting—The rosettes of both events        were larger than controls    -   Plant height—No observable differences between experimentals and        controls    -   Fertility (silique number and seed fill)—No observable        differences between experimentals and controls.

Example 4 Lead 17; ME04249; Ceres cDNA 13495746; clone 37288

Ten independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation as per standardprotocol. Two events showing the most advantageous T₁ phenotypes (plantswith larger rosettes, more leaves and thick inflorescences) were chosenfor evaluation in the T₂ generation. The T₂ growth conditions follow theabove T₁ protocol. The experimental design differs from the T₁ plantingin that each T₂ plant is contained within its own pot, and no herbicideselection is used. All pots for each T₂ event are contained within thesame flat and the plants are randomly distributed within each flat. Thecontrols for each set of measurements are the segregating progeny of thegiven T₁ event which do not contain the T-DNA (internal controls). Allanalyses are done via soil-based experiments under long day lightconditions (16 hours) in the Ceres.

Five of the 10 events produced larger rosettes with more leaves andthicker inflorescences compared to the controls. These plants were alsodelayed in flowering time but exhibited normal fertility. Four otherevents were smaller and/or had fertility defects (Table 9). Thetransgenic “control” was a set of plants expressing a different35S::cDNA and which were indistinguishable from the untransformed WSwildtype.

TABLE 9 Qualitative phenotypes observed in T₁ events containing35S::cDNA 13495746 (highlighted events were chosen for T₂ evaluation)

Events ME04249-03 and ME04249-07 were evaluated in greater detail in theT₂ generation. Eighteen individuals were sown and observed for eachevent. The transgenic plants showed increased primary inflorescencethickness, increased number of rosette leaves, a larger rosette, anddelay of flowering time to a 0.05 level of statistical significance(Table 10). ME04249-07 was also significantly taller than the controls.All plants noted in Table 10 were segregating progeny of the T₁ eventwhich had been confirmed to contain the transgene under test. However,all plants noted in the table as -03 or -07 Control were T₂ plants whichdid not contain the transgene under test (internal controls).

In event ME04249-03 all 11 transgene-containing plants exhibited thebeneficial phenotypes. Event ME04249-07 had 5 transgene-containingplants which exhibited the beneficial phenotype and 2transgene-containing plants which appeared wild-type. Our statisticalanalyses compared the internal controls to those plants with thebeneficial phenotype which contained the transgene; alltransgene-containing (as confirmed by PCR) but wild-type appearingplants were omitted from the statistical analyses in Table 10.

Segregation frequencies of the transgene under test suggest that eachevent contains a single insert, as calculated by a Chi-square test.

TABLE 10 Quantitative phenotypes observed in 35S::cDNA 13495746 T₂events Number of Rosette Number Height Primary InflorescenceEvent/Control Observations Area (mm²) of Leaves (cm) Thickness (inches)Days to Bolt ME04249-03 11 3718.4* 11.0* 55.9 0.060* 22.7* -03 Control 71094.7 7.0 52.0 0.045 18.3 ME04249-07 5 4336.2* 12.8* 79.6* 0.055* 23.6*-07 Control 11 1787.8 6.8 47.9 0.045 17.4 *significantly different fromcontrol at 0.05 level, via t-test

Summary of Results:

-   -   The ectopic expression of cDNA 13495746 with a strong        constitutive promoter (35S) results in plants with thicker        inflorescences, a larger rosette, and more rosette leaves.    -   In the events studied, the increase in plant size seen by this        expression is accompanied by a delay in flowering time, but no        reduction in fertility.

As a result, this gene/protein is especially useful for controlling therate of cell divisions in meristems without disturbing overall plantmorphology. It can be used in crops with an appropriate promoter toregulate size and growth rate of many individual organs. The protein maybe used for creating sturdier stems in corn and preventing “snap”.Increased vegetative biomass gives an improved source:sink ratio andimproved fixation of carbon to sucrose and starch.

Example 5 Lead 50; ME07495-03; ME07495-05; Ceres cDNA 12420535; clone2835

Eight independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation as per standardprotocol. Two events showing the most advantageous T₁ phenotypes werechosen for evaluation in the T₂ generation. The T₂ growth conditionsfollow the above T₁ protocol. The experimental design differs from theT₁ planting in that each T₂ plant is contained within its own pot and noherbicide selection is used. All pots for each T₂ event are containedwithin the same flat, and the plants are randomly distributed withineach flat. The controls for each set of measurements are the segregatingprogeny of the given T₁ event which do not contain the T-DNA (internalcontrols). All analyses are done via soil-based experiments under longdaylight conditions (16 hours) in the Ceres greenhouse.

This transgene produced a qualitative increase in overall plantsize/biomass in 6 of 8 events with no fertility defects. These plantsappeared to be delayed in flowering time by several days (Table 11). Thetransgenic “control” was a set of plants expressing a different 35S:cDNAfusion and were indistinguishable from the untransformed Ws wildtype.

TABLE 11 Qualitative phenotypes observed in 35S::cDNA 12420535 T₁ events(all mutant phenotypes were equivalent, so the 2 highlighted events wererandomly chosen for T₂ analyses).

Events ME07495-03 and ME07495-05 were evaluated in greater detail in theT₂ generation. Eighteen individuals for both events were sown andanalyzed. Segregation frequencies of the plants under test suggest thateach event contains a single insert, as calculated by a Chi-square test.

After detailed T₂ analyses, the following observations were determinedregarding the transgenics (results below noted with a “*” arestatistically significant to a 0.05 level or better via t-test unlessotherwise noted):

Flowering time (days to bolt) was approximately 6 days later thancontrols.

Rosette leaf number at bolting was increased by approximately 3-4leaves.

Rosette area was approximately 3.5 times larger than controls.

Height was increased approximately 20% (12 cm).

Primary inflorescence thickness was increased approximately 30%.

Details can be found in Tables 12-13.

TABLE 12 Quantitative phenotypes observed in 35S::cDNA 12420535 T₂plants. Event/ Number of Day to Number of Rosette Height ControlObservations Bolt Leaves Area (mm²) (cm) ME07495-03 13 30.1* 11.7*4757.4* 61*  -03 Control 5 24 7.4 1434.5 46.8 ME07495-05 10 30.9* 11.1*4001.9*  57.1* -05 Control 8 24.4 8.4 1077.8 47.4 *significantlydifferent from control at 0.05 level, via t-test

TABLE 13 Quantitative phenotypes observed in 35S::cDNA 12420535 T₂plants. Number of Primary Inflorescence Number of Event/ControlObservations Thickness (mm) Inflorescences ME07495-03 13 1.464* 6*  -03Control 5 0.996 3.2 ME07495-05 10 1.69* 6   -05 Control 8 1.16  5.25*significantly different from control at 0.05 level, via t-test

Summary of Results:

-   -   The ectopic expression of cDNA 12420535 with a strong        constitutive promoter (35S) results in taller plants with        thicker inflorescences, larger rosettes, and more rosette        leaves.    -   The increase in plant size seen by this expression is        accompanied by a delay in flowering time but no reduction in        fertility.

As a result, this gene/sequence can provide Increased vegetative biomassto give an improved source:sink ratio to improve yield. Tallerinflorescences give the opportunity for more flowers and, therefore,more seeds. The combination of improved biomass and inflorescencestature can give a significant improvement in yield. Thickerinflorescences can prevent “snap” caused by wind, rain, or drought.

Example 6 Lead 58; ME07957-01, 02 and -06; Ceres cDNA 1241656; Clone235672

Ten independently transformed events were selected and evaluated fortheir qualitative phenotype in the T₁ generation as per standardprotocol. Three events showing the most advantageous T₁ phenotypes werechosen for evaluation in the T₂ generation. The T₂ growth conditionsfollow the above T₁ protocol. The experimental design differs from theT₁ planting in that each T₂ plant is contained within its own pot and noherbicide selection is used. All pots for each T₂ event are containedwithin the same flat, and the plants are randomly distributed withineach flat. The controls for each set of measurements are the segregatingprogeny of the given T₁ event which do not contain the T-DNA (internalcontrols). All analyses are done via soil-based experiments under longdaylight conditions (16 hours) in the greenhouse.

This transgene produced an increase in overall plant size/biomass in 9of 10 events with no fertility defects. These plants appeared to bedelayed in flowering time by several days (Table 14). The transgenic“control” was a set of plants expressing a different 35S::cDNA fusionand were indistinguishable from the untransformed Ws wildtype (thismethod of scoring phenotypes is typical for our large-scalemorphological phenotyping project).

TABLE 14 Qualitative phenotypes observed in 35S::cDNA 12414656 T₁ events(all mutant phenotypes were equivalent, so the 3 highlighted events wererandomly chosen for T₂ analyses).

Events ME07957-01, ME07957-02, and ME07957-06 were evaluated in greaterdetail in the T₂ generation. Eighteen individuals for each event weresown and analyzed. Segregation frequencies of the plants under testsuggest that each event contains a single insert, as calculated by aChi-square test.

After detailed T₂ analyses, we determined the following regarding thetransgenics (results below noted with a “*” are statisticallysignificant to a 0.05 level or better via t-test unless otherwisenoted):

-   -   Flowering time (days to bolt) was approximately 10 days later        than controls.    -   Rosette leaf number at bolting was increased by approximately 10        leaves.    -   Rosette area was approximately 4.7 times larger than controls.    -   Height was increased approximately 20% (12 cm).    -   Primary inflorescence thickness was increased approximately 12%.    -   Inflorescence number increased by approximately 2.1 more        branches than controls.

Details can be found in Tables 15-16.

TABLE 15 Quantitative phenotypes observed in 35S::cDNA 12414656 T₂plants. Event/ Number of Day to Number of Rosette Height ControlObservations Bolt Leaves Area (mm²) (cm) ME07957-01 12 31.8* 17.1*9086.5* 59.0* -01 Control 6 21.5 8.0 1670.9 45.5 ME07957-02 10 31.0*16.4* 8404.7* 59.7* -02 Control 8 21.4 7.6 2022.8 47.3 ME07957-06 1232.0* 18.6* 10312.9* 60.1* -06 Control 6 21.5 7.8 2278.6 50.5*significantly different from control at 0.05 level, via t-test

TABLE 16 Quantitative phenotypes observed in 35S::cDNA 12414656 T₂plants. Number of Primary Inflorescence Number of Event/ControlObservations Thickness (mm) Inflorescences ME07957-01 12 1.50* 8.2* -01Control 6 1.34 6.0 ME07957-02 10 1.57* 8.3* -02 Control 8 1.42 5.8ME07957-06 12 1.63* 7.3* -06 Control 6 1.40 5.7 *significantly differentfrom control at 0.05 level, via t-test

Summary of Results:

-   -   The mis-expression of cDNA 12414656 with a strong constitutive        promoter (35S) results in taller plants with thicker        inflorescences, larger rosettes, and more rosette leaves.    -   The increase in plant size seen by this expression is        accompanied by a delay in flowering time but no reduction in        fertility.    -   Since TxP experiments show that this gene is up-regulated under        drought and SA application, this gene may be useful for stress        tolerance.

As a result, this cDNA 12414656 can be used to increase vegetativebiomass to give an improved source:sink ratio and improve yield. Tallerinflorescences give the opportunity for more flowers and, therefore,more seeds. The combination of improved biomass and inflorescencestature can give a significant improvement in yield. Thickerinflorescences can prevent “snap” caused by wind, rain, or drought. Thisgene/protein can be especially useful for controlling the number/rate ofcell divisions in meristems without disturbing overall plant morphologyand could be developed in crops with an appropriate promoter to regulatesize and growth rate of many individual organs.

Example 7 Determination of Functional Homolog Sequences

The “Lead” sequences described in above Examples 1-6 are utilized toidentify functional homologs of the lead sequences and, together withthose sequences, are utilized to determine a consensus sequence for agiven group of lead and functional homolog sequences.

A subject sequence is considered a functional homolog of a querysequence if the subject and query sequences encode proteins having asimilar function and/or activity. A process known as Reciprocal BLAST(Rivera et al, Proc. Natl Acad. Sci. USA, 1998, 95:6239-6244) is used toidentify potential functional homolog sequences from databasesconsisting of all available public and proprietary peptide sequences,including NR from NCBI and peptide translations from Ceres clones.

Before starting a Reciprocal BLAST process, a specific query polypeptideis searched against all peptides from its source species using BLAST inorder to identify polypeptides having sequence identity of 80% orgreater to the query polypeptide and an alignment length of 85% orgreater along the shorter sequence in the alignment. The querypolypeptide and any of the aforementioned identified polypeptides aredesignated as a cluster.

The main Reciprocal BLAST process consists of two rounds of BLASTsearches; forward search and reverse search. In the forward search step,a query polypeptide sequence, “polypeptide A,” from source species S^(A)is BLASTed against all protein sequences from a species of interest. Tophits are determined using an E-value cutoff of 10⁻⁵ and an identitycutoff of 35%. Among the top hits, the sequence having the lowestE-value is designated as the best hit, and considered a potentialfunctional homolog. Any other top hit that had a sequence identity of80% or greater to the best hit or to the original query polypeptide isconsidered a potential functional homolog as well. This process isrepeated for all species of interest.

In the reverse search round, the top hits identified in the forwardsearch from all species are used to perform a BLAST search against allprotein or polypeptide sequences from the source species S^(A). A tophit from the forward search that returned a polypeptide from theaforementioned cluster as its best hit is also considered as a potentialfunctional homolog.

Functional homologs are identified by manual inspection of potentialfunctional homolog sequences. Representative functional homologs areshown in FIGS. 1-5. Each Figure represents a grouping of a lead/querysequence aligned with the corresponding identified functional homologsubject sequences. Lead sequences and their corresponding functionalhomolog sequences are aligned to identify conserved amino acids and todetermine a consensus sequence that contains a frequently occurringamino acid residue at particular positions in the aligned sequences, asshown in FIGS. 1-5.

Each consensus sequence then is comprised of the identified and numberedconserved regions or domains, with some of the conserved regions beingseparated by one or more amino acid residues, represented by a dash (-),between conserved regions.

Useful polypeptides of the inventions, therefore, include each of thelead and functional homolog sequences shown in FIGS. 1-5, as well as theconsensus sequences shown in those Figures. The invention alsoencompasses other useful polypeptides constructed based upon theconsensus sequence and the identified conserved regions. Thus, usefulpolypeptides include those which comprise one or more of the numberedconserved regions in each alignment table in an individual Figuredepicted in FIGS. 1-5, wherein the conserved regions may be separated bydashes. Useful polypeptides also include those which comprise all of thenumbered conserved regions in an individual alignment table selectedfrom FIGS. 1-5, alternatively comprising all of the numbered conservedregions in an individual alignment table and in the order as depicted inan individual alignment table selected from FIGS. 1-5. Usefulpolypeptides also include those which comprise all of the numberedconserved regions in an individual alignment table and in the order asdepicted in an individual alignment table selected from FIGS. 1-5,wherein the conserved regions are separated by dashes, wherein each dashbetween two adjacent conserved regions is comprised of the amino acidsdepicted in the alignment table for lead and/or functional homologsequences at the positions which define the particular dash. Such dashesin the consensus sequence can be of a length ranging from length of thesmallest number of dashes in one of the aligned sequences up to thelength of the highest number of dashes in one of the aligned sequences.

Such useful polypeptides can also have a length (a total number of aminoacid residues) equal to the length identified for a consensus sequenceor of a length ranging from the shortest to the longest sequence in anygiven family of lead and functional homolog sequences identified in anindividual alignment table selected from FIGS. 1-5.

The present invention further encompasses nucleotides that encode theabove described polypeptides, as well as the complements thereof, andincluding alternatives thereof based upon the degeneracy of the geneticcode.

The invention being thus described, it will be apparent to one ofordinary skill in the art that various modifications of the materialsand methods for practicing the invention can be made. Such modificationsare to be considered within the scope of the invention as defined by thefollowing claims.

Each of the references from the patent and periodical literature citedherein is hereby expressly incorporated in its entirety by suchcitation.

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1. An isolated nucleic acid molecule comprising: (a) a nucleotidesequence encoding an amino acid sequence that is at least 85% identicalto any one of SEQ ID Nos. 2, 10, 17, 22, 27 and 4, respectively; (b) anucleotide sequence that is complementary to any one of the nucleotidesequences according to paragraph (a); (c) a nucleotide sequenceaccording to any one of SEQ ID Nos. 1, 9, 16, 21, 26 and 3; (d) anucleotide sequence that is an interfering RNA to the nucleotidesequence according to paragraph (a); (e) a nucleotide sequence able toform a hybridized nucleic acid duplex with the nucleic acid according toany one of paragraphs (a)-(d) at a temperature from about 40° C. toabout 48° C. below a melting temperature of the hybridized nucleic acidduplex; (f) a nucleotide sequence encoding any one of the amino acidsequences identified as SEQ ID Nos. 2, 10, 17, 22, 27 and 4; or (g) anucleotide sequence encoding any one of the sequences in FIGS. 1-5.
 2. Avector, comprising: a) a first nucleic acid having a regulatory regionencoding a plant transcription and/or translation signal; and a secondnucleic acid having a nucleotide sequence according to any one thenucleotide sequences of claim 1, wherein said first and second nucleicacids are operably linked.
 3. A method of modulating plant size,modulating vegetative growth, modulating plant architecture and/ormodulating the plant biomass, said method comprising introducing into aplant cell an isolated nucleic acid comprising a nucleotide sequenceselected from the group consisting of: (a) a nucleotide sequenceencoding an amino acid sequence that is at least 85% identical to SEQ IDNos. 2, 10, 17, 22, 27 and 4; (b) a nucleotide sequence that iscomplementary to any one of the nucleotide sequences according toparagraph (a); (c) a nucleotide sequence according to any one of SEQ IDNos. 1, 9, 16, 21, 26 and 3; (d) a nucleotide sequence that is aninterfering RNA to the nucleotide sequence according to paragraph (a);(e) a nucleotide sequence able to form a hybridized nucleic acid duplexwith the nucleic acid according to any one of paragraphs (a)-(d) at atemperature from about 40° C. to about 48° C. below a meltingtemperature of the hybridized nucleic acid duplex; (f) a nucleotidesequence encoding any one of the amino acid sequences identified as SEQID Nos. 2, 10, 17, 22, 27 and 4; or (g) a nucleotide sequence encodingany one of the sequences in FIGS. 1-5, wherein said plant produced fromsaid plant cell has modulated plant size, modulated vegetative growth,modulated plant architecture and/or modulated biomass as compared to thecorresponding level in tissue of a control plant that does not comprisesaid nucleic acid.
 4. The method according to claim 3, wherein saidsequence comprises one or more of the conserved regions identified inany one of the alignment tables in FIGS. 1-5.
 5. The method according toclaim 4, wherein said sequence comprises all of the conserved regionsidentified in any one of the sequences shown in the alignment tables inFIGS. 1-5.
 6. The method according to claim 5, wherein said sequencecomprises all of the conserved regions and in the order identified inany one of the sequences shown in the alignment tables in FIGS. 1-5. 7.The method according to claim 6, wherein said conserved regions areseparated by gaps comprised of one or more amino acid residues.
 8. Themethod according to claim 7, wherein each of said gaps is comprised ofone or more amino acids consisting in number and kind of the amino acidsdepicted in the alignment table for the lead and/or functional homologsequences at the corresponding positions that define said gap.
 9. Themethod according to claim 8, wherein said sequence has a length in termsof total number of amino acids that is equal to the length identifiedfor a sequence in one of FIGS. 1-5, or equal to a length ranging fromthe shortest to the longest sequence in any individual alignment tablein any one of FIGS. 1-5.
 10. The method of claim 3, wherein saiddifference is an increase in the level of plant size, vegetative growth,organ number and/or biomass.
 11. The method of claim 3, wherein saidisolated nucleic acid is operably linked to a regulatory region.
 12. Themethod of claim 11, wherein said regulatory region is a promoterselected from the group consisting of YP0092 (SEQ ID NO:68), PT0676 (SEQID NO: 42), PT0708 (SEQ ID NO:47), PT0613 (SEQ ID NO:35), PT0672 (SEQ IDNO: 41), PT0678 (SEQ ID NO:43), PT0688 (SEQ ID NO45:), PT0837 (SEQ IDNO:54), the napin promoter, the Arcelin-5 promoter, the phaseolin genepromoter, the soybean trypsin inhibitor promoter, the ACP promoter, thestearoyl-ACP desaturase gene, the soybean α′ subunit of β-conglycininpromoter, the oleosin promoter, the 15 kD zein promoter, the 16 kD zeinpromoter, the 19 kD zein promoter, the 22 kD zein promoter, the 27 kDzein promoter, the Osgt-1 promoter, the beta-amylase gene promoter, andthe barley hordein gene promoter.
 13. The method of claim 11, whereinsaid regulatory region is a promoter selected from the group consistingof p326 (SEQ ID NO:106), YP0144 (SEQ ID NO:85), YP0190 (SEQ ID NO:89),p13879 (SEQ ID NO:105), YP0050 (SEQ ID NO:65), p32449 (SEQ ID NO:107),YP0158 (SEQ ID NO:87), YP0214 (SEQ ID NO:91), YP0380 (SEQ ID NO:100),PT0848 (SEQ ID NO:56), and PT0633 (SEQ ID NO:37), the cauliflower mosaicvirus (CaMV) 35S promoter, the mannopine synthase (MAS) promoter, the 1′or 2′ promoters derived from T-DNA of Agrobacterium tumefaciens, thefigwort mosaic virus 34S promoter, actin promoters such as the riceactin promoter, and ubiquitin promoters such as the maize ubiquitin-1promoter.
 14. The method of claim 11, wherein said regulatory region isa promoter selected from the group consisting ofribulose-1,5-bisphosphate carboxylase (RbcS) promoters such as the RbcSpromoter from eastern larch (Larix laricina), the pine cab6 promoter,the Cab-1 gene promoter from wheat, the CAB-1 promoter from spinach, thecab1R promoter from rice, the pyruvate orthophosphate dikinase (PPDK)promoter from corn, the tobacco Lhcb1*2 promoter, the Arabidopsisthaliana SUC2 sucrose-H+ symporter promoter, and thylakoid membraneprotein promoters from spinach (psaD, psaF, psaE, PC, FNR, atpC, atpD,cab, rbcS, PT0535 (SEQ ID NO:33), PT0668 (SEQ ID NO:32), PT0886 (SEQ IDNO:59), PR0924 (SEQ ID NO:108), YP0144 (SEQ ID NO:85), YP0380 (SEQ IDNO:100) and PT0585 (SEQ ID NO:34).
 15. A plant cell comprising anisolated nucleic acid comprising a nucleotide sequence selected from thegroup consisting of: (a) a nucleotide sequence encoding an amino acidsequence that is at least 85% identical to any one of SEQ ID Nos. 2, 10,17, 22, 27 and 4; (b) a nucleotide sequence that is complementary to anyone of the nucleotide sequences according to paragraph (a); (c) anucleotide sequence according to any one of SEQ ID Nos. 1, 9, 16, 21, 26and 3; (d) a nucleotide sequence that is an interfering RNA to thenucleotide sequence according to paragraph (a); (e) a nucleotidesequence able to form a hybridized nucleic acid duplex with the nucleicacid according to any one of paragraphs (a)-(d) at a temperature fromabout 40° C. to about 48° C. below a melting temperature of thehybridized nucleic acid duplex; (f) a nucleotide sequence encoding anyone of the amino acid sequences identified as SEQ ID Nos. 2, 10, 17, 22,27; or (g) a nucleotide sequence encoding any one of the sequences inFIGS. 1-5.
 16. A transgenic plant comprising the plant cell of claim 15.17. Progeny of the plant of claim 16, wherein said progeny has modulatedplant size, modulated vegetative growth, modulated plant architectureand/or modulated biomass as compared to the corresponding level intissue of a control plant that does not comprise said nucleic acid. 18.Seed from a transgenic plant according to claim
 16. 19. Vegetativetissue from a transgenic plant according to claim
 16. 20. A food productcomprising vegetative tissue from a transgenic plant according to claim16.
 21. A feed product comprising vegetative tissue from a transgenicplant according to claim
 16. 22. A product comprising vegetative tissuefrom a transgenic plant according to claim 16 used for the conversioninto fuel or chemical feedstock.
 23. A method for detecting a nucleicacid in a sample, comprising: providing an isolated nucleic acidaccording to claim 1; contacting said isolated nucleic acid with asample under conditions that permit a comparison of the nucleotidesequence of the isolated nucleic acid with a nucleotide sequence ofnucleic acid in the sample; and analyzing the comparison.
 24. A methodfor promoting increased biomass in a plant, comprising: (a) transforminga plant with a nucleic acid molecule comprising a nucleotide sequenceencoding any one of the sequences in any one of FIGS. 1-5; and (b)expressing said nucleotide sequence in said transformed plant, wherebysaid transformed plant has an increased biomass as compared to a plantthat has not been transformed with said nucleotide sequence.
 25. Amethod for modulating the biomass of a plant, said method comprisingaltering the level of expression in said plant of a nucleic acidmolecule according to claim 1.